1.Molecular Mechanism And Biology Function Of Proteinase Activated Receptor 2 （PAR₂）-induced YAP Activation 2.Expression Of MicroRNA-181b In Colitis-associated Colonic Carcinogenesis

Partâ… Molecular Mechanism and Biology Function of Proteinase activated Receptor 2 （PAR₂）-induced YAP activationChronic intestinal inflammation （eg. Inflammatory bowel diseases）, recurrently causes mucosal damage followed by repair and regeneration, and also is one of the high risk factors of colorectal cancers. Colitis-associated carcinogenesis is quite complex and involves multiple pathway. Proteinase activated Receptor 2, a G-protein coupled receptor, has been reported to mediate inflammatory response,. PAR₂ was over expression in IBD animal models, however, it is unclear whether PAR₂ is required in inflammation-induced intestinal epithelial regeneration. YAP is the most important element of Hippo pathway, which plays critical role in the processes of tissue regeneration and homeostasis. Here we studied the molecular mechanism by which PAR₂ activated YAP and the role of PAR₂-induced YAP activation in co lonic regeneration after inflammation-induced damage.Firstly, our study demonstrated that activation of PAR₂ signaling induced nuclear localization of YAP and increased its transcriptional activity. Stable knock down of PAR₂ not only decreased stability of YAP but also inhibited transcriptional activity of TEAD. Western Blot showed that stimulation of PAR₂ induced YAP de-phosphorylation at Ser 127 and Ser 397, but this was independent of Hippo pathway. To test whether phosphatase meditate PAR₂-related phosphorylation of YAP, chemical inhibitor OA （Okadaic acid） and siRNA against phosphatase 1（PP1） were used to selectively inhibit the activity or expression of PP1 It was found that both OA and siRNA blocked PAR₂ induced YAP de-phosphorylation. Furthermore, CoIP assay showed that activation of PAR₂ significantly increased the interaction between PP1 and YAP. In addition to the dephosphorylation of YAP at ser 127 and ser397, PAR₂ also increased YAP phosphorylation at tyrosine Y357 which was completely abolished by the inhibitor of SRC family dasatinib. To identify the specific tyrosine kinase mediated PAR₂-induced YAP phosphorylation at tyrosine Y357, FYN was chosen due to our previous finding that PAR₂ regulated FYN expression. Although knock down of FYN with siRNA dramatically decreased YAP protein expression, PAR₂-induced YAP tyrosine phosphorylation was not affected. It was worth mentioning that siRNA targeted GAB2 （GRB2-associated-binding protein 2）, which is an important docking protein related to receptor or nonreceptor tyrosine kinase, not only blocked the de-phosphorylation of YAP at Ser 127 but also decreased levels of PP1 and FYN. To test whether PAR₂-induced activation of YAP play important role in vivo, acute colitis was induced by dextran sulfate sodium （DSS） in C57BL wild type or PAR₂-/-mouse. Compared with wild type mice, crypt number, expression and nuclear accumulation of YAP protein in colon tissue dramatically decreased in PAR₂-/-mice 2 and 4 days after the withdrawal of DSS. These indicated that PAR₂ signaling was involved in the regeneration process after colonic damage induced by DSS and YAP might mediate this process.In summary, PAR₂ promoted nuclear accumulation, protein stability and transcriptional activity of YAP through dephosphorylation and phosphorylation dependent mechanisms. In vivo, PAR₂-associated YAP activation might be involved in intestinal epithelial repair and regeneration after inflammatory damage.Part â…¡Expression of miR-181b in Colitis-associated Colonic CarcinogenesismiRNAs as single-stranded small non-coding RNAs promoted tumorigenesis by inhibiting the expression of oncogene or tumor suppressor. miRNA-181b significantly over-expressed in colorectal cancer. however, it is unknown whether miRNA-181b participate in Colitis-associated Colonic Carcinogenesis progression. We studied the expression change of miR-181b in colitis-associated colonic carcinogenesis model in this part.To study it, Colitis-associated colon cancer model was induced by combination of azoxymethane （AOM） and dextran sulfate sodium （DSS） in mice. The expression of miR-181b at different time in colon tissue was measured by real time fluorescent quantitative PCR. The expression of miR-181b was gradually elevated during the development of colitis-associated colon cancer. However, DSS or AOM alone did not change expression of miR-181b. Genome microarray combined with miRNA target gene prediction softwares showed that Ipmk, E2f5, K1f6, Bai3, Hic2, Prkcd may be the target genes of miR-181b. Real-time PCR showed that expressions of Ipmk, Bai3, Hic2 and Prkcd were significantly decrased in colitis-associated colon cancer than in control group.To sum up, The expression of miR-181b was gradually elevated during the development of colitis-associated colon cancer model. this indicated miR-181b might be related to colitis-associated carcinogenesis.